Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections

Diabetes mellitus (DM) patients frequently develop diabetic foot ulcers (DFU) which are generally infected by a community of microorganisms, mainly Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria exhibit a multi-drug resistance profile and biofilm-forming ability which represent a hurdle in the treatment of diabetic foot infections (DFI). We aimed to evaluate the potential of Nisin Z, an antimicrobial peptide (AMP), as an alternative treatment for severe DFI. Nisin Z shows antibacterial activity against Gram-positive and Gram-negative bacteria and an increased antibacterial effect against Gram-negatives when added to EDTA. As such, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), and Minimum Biofilm Eradication Concentration (MBEC) were determined for Nisin Z, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA incorporated into guar gum, in order to test its efficacy against S. aureus and P. aeruginosa isolated from the same DFU. Results showed that Nisin Z added to the chelation agent EDTA displayed higher antibacterial and bacteriostatic efficacy against mono and dual co-cultures of S. aureus and P. aeruginosa, and higher antibiofilm efficiency against monocultures. Nisin Z was moderately cytotoxic at 200 µg/mL. Prospect in vivo studies are needed to confirm the potential of Nisin Z supplemented with EDTA to be used as a complement to conventional antibiotic therapy for severe DFI.


Introduction
Diabetes mellitus (DM) is a lifelong chronic metabolic disease that affects about 537 million people worldwide, and the prevalence of which is increasing [1]. Approximately 30% of patients with diabetes develop diabetic foot ulcers (DFU) [2], because of a complex interaction of several pathophysiological factors affecting the vascular and immune systems and causing nerve damage, often affecting the legs and feet [3]. Around 40% of patients with DFU experience a recurrence within one year after the ulcer has healed, while nearly 60% suffer recurrence within three years, and 65% within five years [4]. The severe loss of skin protective barrier creates an opportunity for tissue colonization by bacteria, and 50% of diabetic patients with a DFU are estimated to develop diabetic foot infections (DFI) in their lifetime [2], which can be colonized by a polymicrobial community of opportunistic microorganisms [5]. DFI usually become chronic, resulting in a decrease in life quality, repeated hospitalizations, nontraumatic lower extremity amputation, higher morbidity, and premature mortality [6]. and P. aeruginosa DFI strains, previously characterized by us [33][34][35][36], were assessed. Nisin Z cytotoxicity was also evaluated.

Strains and Cultural Conditions
Biofilm-producing DFI strains S. aureus Z25.2 and P. aeruginosa Z25.1, co-isolated from the same diabetic foot ulcer in an epidemiological study on DFI microbiota conducted by us and fully characterized [33][34][35][36], were used. Reference strains S. aureus ATCC 29,213 and P. aeruginosa ATCC 27,853 were used as control strains. Before testing, strains were inoculated in Brain Heart Infusion (BHI) agar (VWR, Leuven, Belgium) and incubated at 37 • C, for 24 h. Bacterial suspensions in a sterile saline solution with 10 8 CFU/mL were prepared from plate cultures and diluted in fresh BHI broth (VWR, Leuven, Belgium) to 10 7 and 10 6 CFU/mL.
A guar gum gel of 1.5% (w/v) was prepared by dissolving 0.75 g of guar gum (Sigma-Aldrich, Darmstadt, Germany) in 50 mL of sterile distilled water and heat sterilized by autoclave. An EDTA disodium salt (Sigma-Aldrich, Darmstadt, Germany) stock solution of 64 mg/mL was prepared by dissolving 6.4 g of EDTA in 100 mL of sterile distilled water and filtered.
MIC value of EDTA and Nisin Z solutions (tested concentrations ranging from 0.1 to 400 µg/mL) were determined by microtitre broth dilution method [37]. MIC was carried out in 96-well flat-bottomed polystyrene microtitre plates (VWR, Radnor, PA, USA). Briefly, 50 µL of each Nisin Z solution was inoculated onto each well of a 96-well plate, except for the positive (200 µL of bacterial suspension) and negative control wells (200 µL of broth medium). Then, all wells, except for positive and negative controls, were inoculated with 150 µL of the 10 7 CFU/mL suspensions, as follows: plates 1/2/3-S. aureus suspension (DFI and reference strain); 4/5/6-P. aeruginosa suspension (DFI and reference strain); 7/8/9-S. aureus + P. aeruginosa dual-suspension (1:1) (dual DFI and dual reference strains). Microplates were incubated overnight at 37 • C, and MIC determined as the lowest concentration of each Nisin Z solution that visually inhibits microbial growth.
MBC value was determined by inoculating 3 µL of the suspensions of the wells with no visual bacterial growth onto BHI agar plates, incubated at 37 • C, for 24 h. MBC was determined as the lowest concentration of each Nisin Z solution at which no colonies were formed.
Experiments were conducted in triplicate, and independent replicates were performed at least three times on different days. For each strain, nine results were obtained and analyzed.

Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC)
MBIC and MBEC of Nisin Z, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA (0.4%) + guar gum gel 0.75% (w/v), were determined using a modified version of the Calgary Biofilm Pin Lid Device [38]. Briefly, bacterial suspensions were prepared as before and diluted to 10 6 CFU/mL in Tryptic Soy Broth medium (TSB) (VWR, Brussels, Belgium) supplemented with 0.25% (w/v) glucose (Merck, NJ, USA). Then, 200 µL of suspensions were placed in 96-well flat-bottomed polystyrene microtitre plates, covered with 96-peg polystyrene lids (Nunc, Thermo Fisher Scientific, Roskilde, Denmark) and statically incubated overnight at 37 • C to allow biofilm formation on pegs. Nine plates with DFI or reference strains were prepared for each determination, as in the previous MIC and MBC protocols.
For MBEC determination, peg lids were rinsed in PBS, placed in new microplates with 200 µL of fresh TSB with 0.25% glucose, and incubated in an ultrasound bath (1 min, 50 Hz) to disperse biofilms from the pegs' surface. Then, peg lids were discarded, microplates covered with normal lids and incubated for 24 h, at 37 • C. MBEC value was determined as the minimum concentration of each Nisin Z solution causing no visual growth relative to the positive control [15].
Experiments were conducted in triplicate, and independent replicates were performed at least three times on different days. For each strain, nine results were obtained and analyzed.

Cytotoxicity Assay
Cytotoxicity assay was performed as previously described [32]. Adult Human primary adherent Epidermal Keratinocytes (HEKa) (PCS-200-011, ATCC, Manassas, VA, USA) were cultured according to the manufacturer's instructions to assess the cytotoxic potential of each Nisin Z solution against eukaryotic cells.
Cell viability was determined using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay (MTT assay) kit (ab211091, Abcam, Cambridge, UK), with small changes to the manufacturer's instructions. Briefly, growth medium was removed from all wells, and growth medium and MTT reagent (1:1) were added into each well. Cells were then incubated at 37 • C for 3 h, after which 150 µL of MTT solvent was added into each well to dissolve the formazan crystals. Microplates were wrapped in foil and agitated on an orbital shaker for 15 min at room temperature. Cell viability was evaluated using a microplate reader (BGM LABTECH, Ortenberg, Germany) to measure optical density at a wavelength of 584 nm. Blank control was the growth medium without cells. Cell viability was calculated as a percentage of the untreated control (growth media plus HEKa cells), which was assumed to be 100% viable.
Experiments were conducted in triplicate, and independent replicates were performed at least three times on different days. For each strain, nine results were obtained and analyzed.

Statistical Analysis
Data were analyzed using GraphPad Prism software for Windows version 9.4.1 (graph Pad, USA) and shown as mean ± standard error of mean (SEM). One-way ANOVA (p < 0.0001) and Tukey's multiple comparisons test were performed. MIC, MBC, MBIC and MBEC analysis were conducted separately. In each of them, the differences between Nisin Z solutions were analyzed collectively for S. aureus Z.25.2, P. aeruginosa Z.25.1 and dual DFI co-culture. Regarding the DFI and control strains comparisons, the differences between Nisin Z solutions were analyzed for each DFI and control pair.

MIC and MBC
MIC and MBC of EDTA were evaluated and showed no inhibitory effect in any tested concentration (250 to 8000 µg/mL) towards planktonic DFI and control strains. EDTA exhibited a MIC and MBC mean value of >8000 ± 0 µg/mL. Therefore, a final concentration of EDTA of 4000 of µg/mL was chosen to be added to Nisin Z solution. S. aureus Z25.2 was susceptible to low Nisin Z concentrations, but P. aeruginosa Z25.1 and dual DFI co-cultures were resistant to the antimicrobial peptide (MIC > 400 µg/mL). Nisin Z MIC towards S. aureus Z25.2 ranged from 5 to 10 µg/mL, with a mean value of 6.11 ± 2.2 µg/mL, and MBC values were two to 2.5-fold higher than MIC (MBC mean of 18.8 ± 6.9 µg/mL, ranging from 10 to 25 µg/mL). P. aeruginosa Z25.1 and the dual DFI co-culture were not affected by Nisin Z (MIC and MBC higher than 400 µg/mL) (Figure 1a,b).
When EDTA (0.4%) was added to Nisin Z, all suspensions were susceptible at low MIC values, with these results being statistically different when compared to Nisin Z MIC towards S. aureus Z.25.2 ( Figure 1a). In addition, MBC values decreased and remained under 200 µg/mL (Figure 1b). Nisin Z MIC regarding S. aureus Z25.2 ranged between 0.25 and 2.5 µg/mL, with a mean value of 1.0 ± 1.1 µg/mL. MBC was four-to six-fold higher, with a mean of 8.6 ± 5.3 µg/mL, ranging between 1 and 15 µg/mL.
Nisin Z MIC regarding P. aeruginosa Z25.1 showed a mean value of 2.5 ± 0 µg/mL. MBC was 40 to 80-fold higher, with a mean value of 150.0 ± 53.5 µg/mL, ranging from 100 to 200 µg/mL. Nisin Z MIC regarding dual DFI co-culture showed a mean value of 2.5 ± 0 µg/mL, and MBC was 40-fold higher, with a mean value of 100.0 ± 0.
After the addition of guar gum, only dual co-cultures were evaluated because it was anticipated that MIC and MBC values regarding DFI monocultures of P. aeruginosa Z.25.1 and S. aureus Z.25.2 would be similar than those for Nisin Z + EDTA (0.4%), and priority was given to the dual co-culture, as the more severe forms of DFI are commonly polymicrobial. The addition of guar gum did not statistically change Nisin Z MIC (2.5 µg/mL) regarding dual DFI co-cultures (Figure 1a), but it changed (p < 0.05) the MBC mean value to 250.0 ± 92.6 µg/mL (200 to 400 µg/mL) (Figure 1b). When EDTA (0.4%) was added to Nisin Z, all suspensions were susceptible at low MIC values, with these results being statistically different when compared to Nisin Z MIC towards S. aureus Z.25.2 (Figure 1a). In addition, MBC values decreased and remained under 200 µg/mL (Figure 1b). Nisin Z MIC regarding S. aureus Z25.2 ranged between 0.25 and 2.5 µg/mL, with a mean value of 1.0 ± 1.1 µg/mL. MBC was four-to six-fold higher, with a mean of 8.6 ± 5.3 µg/mL, ranging between 1 and 15 µg/mL. Nisin Z MIC regarding P. aeruginosa Z25.1 showed a mean value of 2.5 ± 0 µg/mL. MBC was 40 to 80-fold higher, with a mean value of 150.0 ± 53.5 µg/mL, ranging from 100 to 200 µg/mL. Nisin Z MIC regarding dual DFI co-culture showed a mean value of 2.5 ± 0 µg/mL, and MBC was 40-fold higher, with a mean value of 100.0 ± 0.
After the addition of guar gum, only dual co-cultures were evaluated because it was anticipated that MIC and MBC values regarding DFI monocultures of P. aeruginosa Z.25.1 and S. aureus Z.25.2 would be similar than those for Nisin Z + EDTA (0.4%), and priority was given to the dual co-culture, as the more severe forms of DFI are commonly polymicrobial. The addition of guar gum did not statistically change Nisin Z MIC (2.5 µg/mL) . Data shown as means ± SEM; each group value is an average of mostly nine independent measurements. ns not significant: *** p < 0.001; **** p < 0.0001; One-way ANOVA, Tukey's multiple comparison test.
Comparing the results for S. aureus Z.25.2 to those for the reference strain, only the difference in the Nisin Z MBC value was not significantly different. Comparing the results for P. aeruginosa Z.25.1 to those for the reference strain, only Nisin Z + EDTA (0.4%) MIC value was significantly different. Comparing the results from the dual DFI co-culture to those from the dual control, only Nisin Z + EDTA (0.4%) MIC and MBC values were significantly different (Table 1).

MBIC and MBEC
The biofilm mode of growth of the infecting organisms can impair DFI healing, since biofilm can resist antibiotic concentrations 10 to 10,000 times higher than those needed to eliminate planktonic forms [39]. Therefore, to evaluate MBIC and MBEC, Nisin Z was tested using concentrations up to 1250 µg/mL. As P. aeruginosa Z25.1 and dual DFI cocultures were resistant to Nisin Z, it was assumed that they would maintain their resistant phenotype, therefore Nisin Z MBIC and MBEC towards these biofilms were not evaluated.
S. aureus Z.25.2 biofilm was susceptible to Nisin Z solutions at low concentrations, with MBIC ranging from 5 to 10 µg/mL, with a mean value of 6.7 ± 2.5 µg/mL, and MBEC ranging from 200 to 400 µg/mL, with a mean value of 366.7 ± 81.6 µg/mL.
The addition of EDTA (0.4%), and of EDTA (0.4%) + guar gum gel 0.75% (w/v) to Nisin Z render these solutions cytotoxic in all concentrations tested, with cell viability ranging from 9.2% to 33.1% and from 4.6% to 17.4%, respectively (Table 2). Table 2. Cell viability (percentage) of adult human primary adherent epidermal keratinocytes (HEKa) when treated with Nisin Z solutions. Each group value is an average of nine independent measurements.

Discussion
The antibacterial activity of the Nisin Z suspension was improved by the addition of EDTA (0.4%) to this antimicrobial peptide, as S. aureus Z.25.2, P. aeruginosa Z.25.1 and dual DFI co-cultures were considered susceptible at low MIC values. These results are in accordance with a previous study in which the antimicrobial potential of Nisin A was analyzed against 23 S. aureus DFI biofilm producing isolates [31]. In that study, S. aureus Z.25.2 was considered susceptible to Nisin A at MIC = 100 µg/mL, a higher value than the one obtained in the present study (MIC of Nisin Z = 6.1 ± 2.2 µg/mL). The differences between the structure of the two molecules may explain the different results [24,25].
The addition of EDTA (0.4%) to Nisin Z resulted in a suspension with bactericidal potential against P. aeruginosa Z.25.1 and dual DFI co-cultures. Nisin Z solutions, with or without EDTA, had bactericidal potential against S. aureus Z.25.2 with low MBC values. In a previous study, the mean MBC value of Nisin A against S. aureus Z.25.2 was 600 µg/mL [31], a much higher value than the one obtained in the present study (MBC of Nisin Z = 18.8 ± 6.9 µg/mL). These differences observed in MIC and MBC values of Nisin A and Nisin Z against S. aureus Z.25.2 highlight the superior antibacterial efficacy of Nisin Z over Nisin A towards DFI isolates.
Results showed that the addition of guar gum to Nisin + EDTA (0.4%) did not statistically affect the suspension's antibacterial efficacy, but it was detrimental to its bactericidal potential towards co-cultures.
Since antimicrobial agents are usually classified as bactericidal if the MBC is no more four times the MIC value [41], our results showed that Nisin Z is bactericidal against S. aureus, and Nisin Z with EDTA is bacteriostatic against S. aureus Z.25.2 and P. aeruginosa Z.25.1 in monocultures and dual co-cultures. However, Nisin Z + EDTA (0.4%) should be considered a valued AMP to kill planktonic S. aureus, as MBC (four to six-fold) was near the limit value to be considered bactericidal [41].
Nisin Z solutions showed high antibiofilm activity against S. aureus Z.25.2 and P. aeruginosa Z.25.1 biofilms, associated with low MBIC values. The addition of guar gum was beneficial for dual DFI co-cultures because it significantly increased the antibiofilm efficacy to nontoxic Nisin Z levels. Biofilm eradication was only attained towards S. aureus Z.25.2 biofilms, but still in concentrations over 200 µg/mL, and significantly more effective without adding guar gum.
Antibiofilm and eradication efficacy of Nisin Z was demonstrated to be superior to that of Nisin A, since results from a previous study showed that Nisin A MBIC value against S. aureus Z.25.2 was 40 µg mL and MBEC values > 1000 µg mL [31]. The difference in a single amino acid residue at position 27 (asparagine instead of histidine) [24,25], between Nisin A and its closest variant Nisin Z is enough for a higher antibacterial, antibiofilm and eradication potential against DFI S. aureus.
Regarding dual co-cultures, it is recognized that there is a synergy between both P. aeruginosa and S. aureus that may protect them from the inhibitory action of antibiotics [11]. If the synergy effects were stronger than the also known negative interactions of P. aeruginosa against S. aureus, in which P. aeruginosa produces anti-staphylococcal products and proteases causing S. aureus biofilm dispersion and cell lysis [12,42], dual co-cultures would show higher resistance to Nisin Z solutions than monocultures. This was not the case, since Nisin Z MIC, MBC, and MBEC towards dual DFI co-cultures were more similar to those towards P. aeruginosa Z.25.1 and generally higher than those towards S. aureus Z.25.2. Therefore, results suggest that, in vitro, the harmful action of P. aeruginosa against S. aureus overlaps the synergistic effect of being together.
Studies available on the cytotoxicity of Nisin regarding keratinocyte cells are limited, and results depend on the cell type. It was shown that Nisin Z, a naturally occurring variant of Nisin, does not induce apoptosis in human oral keratinocytes [43], and in another study showed that Nisin A concentrations up to 50 µg/mL were non-cytotoxic to HEKa cells [32].
According to [44], generally it can be said that if a compound is non-cytotoxic, then it will be tolerable in vivo. If the compound is moderately cytotoxic, there is a likelihood that the different cell types of an organ can effectively recover from the cytotoxic damage, or the damage will only be minimal [44]. In the present study, Nisin Z at 200 µg/mL was moderately cytotoxic to HEKa cells, meaning that it is within acceptable values for pharmaceutical/medical use; however, in vivo safeness must be further evaluated. Although the addition of EDTA (0.4%) to Nisin Z renders this solution cytotoxic to HEKa cells, EDTA concentration tested was within the range commonly used in therapeutical protocols and considered to be safe [45][46][47]. In fact, according to [47], there are two commercially available wound care products that contain EDTA: RescuDerm (NociPharm, Inc., Toronto, ON, Canada), and Biostep (Smith & Nephew Wound Management, Inc., Largo, FL, USA), both FDA approved. Moreover, EDTA is the medically-accepted FDA-approved treatment for lead poisoning in adults and children [48], and used in the treatment of band keratopathy, a degenerative condition of the cornea [46], through chelation therapy [46,48]. Therefore, EDTA cytotoxicity must be analyzed carefully, as it may be associated with specific in vitro conditions, and probably does not relate to an in vivo cell toxicity, and further analysis is needed. The detected cytotoxicity of Nisin Z with EDTA (0.4%) incorporated into guar gum was most possibly due to the presence of EDTA, as guar gum was considered non-cytotoxic in a previous study in which the incorporation of Nisin A into guar gum gel 0.75% (w/v) had no impact on Nisin A cytotoxicity [32].
Biofilm eradication was not attainable in any tested Nisin Z solution at moderatecytotoxic levels. Nevertheless, this study highlights the antibacterial and antibiofilm activity of Nisin Z supplemented with EDTA against S. aureus and P. aeruginosa, which are ESKAPE pathogens commonly found colonizing diabetic foot ulcers. Further in vivo studies aiming at confirming and characterizing the solution antimicrobial activity towards polymicrobial diabetic foot infections are urgent. We expect that, in the future, Nisin Z supplemented with EDTA would be used as a safe and effective complement to antibiotics for the successful treatment of severe diabetic foot infected ulcers. It would ultimately contribute to stopping the diabetic foot infection from becoming chronic and limiting the quality of patients' life.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.